Developing a novel exenatide-based incretin mimic (αB-Ex): Expression, purification and structural-functional characterization

• Published in: Biochimica et biophysica acta. General subjects Vol. 1866; no. 7; p. 130150
• Main Authors: Ghanbarnezhad, Mohammad Mehdi, Shahsavani, Mohammad Bagher, Mali, Pramod S., Upadhyay, Mansi, Kumar, Ashutosh, Albaghlani, Rawayh Muslim, Niazi, Ali, Yousefi, Reza
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2022
• Subjects: Animals; bioactive properties; blood flow; blood glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2 - drug therapy; drugs; electrospray ionization mass spectrometry; Exenatide - therapeutic use; Exenatide analogue; gel chromatography; glucagon-like peptide 1; humans; Incretin activity; Incretins; insulin secretion; leucine; Liraglutide - therapeutic use; Mass spectroscopy; methionine; Mice; molecular weight; noninsulin-dependent diabetes mellitus; obesity; proteins; Secondary structure; secretin; Type 2 diabetes mellitus (T2DM); αB-Ex hybrid protein; Exenatide analogue; Mass spectroscopy; Secondary structure; Type 2 diabetes mellitus (T2DM); αB-Ex hybrid protein; Incretin activity
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2022.130150

Among the various treatments, GLP-1 receptor agonists (incretin mimics) such as liraglutide and exenatide have been well received in treating type 2 diabetes mellitus (T2DM) and obesity. In this study, an exenatide analogue, in which methionine at position 14 substituted with leucine, was ligated to human αB-crystallin (αB-Cry) and then expressed in the bacterial host cells. In the next step, the exenatide analogue was effectively released from the hybrid protein (αB-Ex) and subsequently purified using gel filtration chromatography. The HPLC and electrospray ionization mass spectrometry (ESI-MS) analyses respectively suggested a high purity (more than 97%) and an accurate molecular mass for the exenatide analogue (4168.22 Da and 835.01, z = 5). Also, the molecular mass of the αB-Ex hybrid protein based on the MALDI-TOF analysis was 24,702.162 Da. The secondary structure assessment by the three spectroscopic methods revealed that exenatide analogue and αB-Ex hybrid protein have an α-helix and a β-sheet rich structure, respectively. Also, according to the results of the DLS analysis, the αB-Ex hybrid protein indicated a high tendency to form large oligomeric structures. The NMR assessment suggested that the hybrid protein exists in its folding state. Both exenatide analogue and the αB-Ex hybrid protein revealed a crucial ability to reduce the blood sugar levels in healthy and diabetic mice. They were also capable of inducing insulin secretion to the bloodstream. Overall, our study introduces the αB-Ex hybrid protein as a novel incretin mimic, exerting its biological activity for a longer period of time. It might also be considered a potential drug candidate in the treatment of T2DM. [Display omitted] •Exenatide is an incretin mimics that used in treatment of T2DM.•αB-Cry was ligated to exenatide at the gene level.•The gene construct was efficiently expressed in Escherichia coli.•Both αB-Ex hybrid protein and exenatide indicated incretin activity.•αB-Ex hybrid protein introduced as a novel incretin mimic.